WO2003020111A2 - Facteur tissulaire circulant presentant un epissage alternatif - Google Patents

Facteur tissulaire circulant presentant un epissage alternatif Download PDF

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WO2003020111A2
WO2003020111A2 PCT/US2002/027533 US0227533W WO03020111A2 WO 2003020111 A2 WO2003020111 A2 WO 2003020111A2 US 0227533 W US0227533 W US 0227533W WO 03020111 A2 WO03020111 A2 WO 03020111A2
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tissue factor
htf
alt
protein
alternatively spliced
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PCT/US2002/027533
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WO2003020111A3 (fr
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Yale Nemerson
Vladimir Bogdanov
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Mount Sinai School Of Medicine Of New York University
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Priority to DE60232733T priority Critical patent/DE60232733D1/de
Priority to AU2002361471A priority patent/AU2002361471A1/en
Priority to AT02797784T priority patent/ATE434404T1/de
Priority to EP02797784A priority patent/EP1429650B1/fr
Publication of WO2003020111A2 publication Critical patent/WO2003020111A2/fr
Publication of WO2003020111A3 publication Critical patent/WO2003020111A3/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/705—Receptors; Cell surface antigens; Cell surface determinants
    • C07K14/70596—Molecules with a "CD"-designation not provided for elsewhere
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07K—PEPTIDES
    • C07K14/00—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • C07K14/435—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof from animals; from humans
    • C07K14/475—Growth factors; Growth regulators
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides

Definitions

  • the present invention is generally in the field of diagnostic and therapeutic reagents, and especially relates to a naturally occurring, circulating soluble alternatively spliced form of human tissue factor (alt-hTF) implicated in thrombotic conditions.
  • alt-hTF human tissue factor
  • Tissue factor is the primary initiator of blood coagulation.
  • TF Tissue factor
  • formation of a TF.FVIIa complex leads to the generation of FXa, thrombin and the deposition of fibrin to limit hemorrhage.
  • FXa FXa
  • thrombin thrombin
  • fibrin fibrin to limit hemorrhage.
  • TF initiates life- threatening intravascular thrombosis in sepsis, atherosclerosis and cancer. More recently, TF has been proposed to play a role in other biological processes, including tumor-associated angiogenesis, metastasis and inflammation. In combination with phospholipid vesicles, TF is used commercially in diagnostic clotting assays.
  • TF is present in only very small amounts on the surface of cells in the body and not in a circulating form.
  • laboratories used thromboplastin, an extract of human brain, placenta or rabbit brain and lung that was previously acetone extracted. The material was lyophilized and resuspended in buffered saline. Until cloned, the structure, exact molecular weight, role of carbohydrate, and relationship with other proteins in humans and other species were not known.
  • tissue factor (263 amino acids in length; GenBank accession number J02931) or human tissue factor modified to yield a truncated or soluble tissue factor (between 218 and 243 amino acids in length) could be expressed in bacteria and mammalian cells.
  • This tissue factor is used in commercial clotting assays.
  • the soluble tissue factor has the advantage that it is easier to produce, purify and resuspend, as compared to the membrane bound form.
  • tissue factor In blood vessels of healthy humans, tissue factor is found primarily in the adventitia and thus physically separated from coagulation factors, which mainly circulate in an inactive form. Following injury, TF is exposed to blood and initiates the coagulation cascade. The resulting fibrin formation is essential for the initial repair of vessel damage to minimize blood-loss. Therefore, TF may be considered to form a hemostatic sheath around blood vessels essential for hemostasis and appears to be essential for life inasmuch as no TF deficiency has been reported and TF knockout mice do not survive beyond the perinatal period.
  • TF also plays a crucial role in pathological situations such as coronary artery disease or deep vein thrombosis (DVT).
  • atherosclerosis is the underlying process leading to pathological disturbances of the arterial wall.
  • the mechanism of venous thrombosis is poorly understood but perhaps blood-borne TF is involved, as reported by Giesen et al., Proc. Natl. Acad. Sci. USA 1999; 96: 2311-2315.
  • Atheromae contain TF as judged by direct bioassay of excised lesions and by immunohistochemistry. Monocytes/macrophages are generally believed to be the major source of this TF although smooth muscle cells near experimental arterial injury contain TF.
  • TF Upon plaque rupture, TF is exposed to flowing blood thereby allowing circulating factor Vll/VIIa to complex with TF.
  • This complex is the catalyst that initiates blood coagulation and thrombosis.
  • the deposition of platelets on a TF-coated disc has been reported to inhibit this surface-bound TF, thus implicating circulating TF as necessary for thrombus propagation (Hathcock and Nemerson, Abstract OC2404, Thrombosis and Haemostasis, Supplement, 2001).
  • soluble hTF may play a role in these disorders as well as normal coagulation, although its role is not clear. It is also not clear what form this protein may have, or its source.
  • Previously reported functional mutants of human tissue factor have been truncation mutants, typically 1, 2, or 3 - 218/219, or mutants engineered to contain amino acid substitutions.
  • One commercially available diagnostic reagent is truncated at residue 243.
  • Alt-hTF A previously unknown circulating form of soluble human tissue factor has been identified. This form of human tissue factor appears to be the result of alternative splicing and is therefore referred to as "alt-hTF.”
  • alt- hTF mRNA was detected in a cell line, HL-60.
  • the cDNA region encoding the entire open reading frame of alt-hTF was cloned.
  • the sequence encoding the alt-hTF mature peptide was expressed in bacteria.
  • Alt-hTF consists of the first 166 amino acids of membrane bound TF, and a 40 amino acid C- terminal region unique to alt-hTF.
  • Alt-hTF is used as a diagnostic and is also a target for compounds to inhibit clotting and to treat disorders associated with elevated TF. It is also useful as a target for antibodies selectively reactive with alt-hTF, to remove it from the circulation for treatment of clotting or other disorders associated with elevated or abnormal levels of TF, including thrombotic conditions, cardiovascular disorders, DVT, DIC, and possibly metastatic cancers.
  • Figure 1 is a schematic of the full-length human tissue factor protein, the alternatively spliced human tissue factor protein, and the regions therein.
  • Figure 2 is a graph of the TF activity [pM factor Xa/min] for the fractions of human plasma centrifuged at 260,000xG, i.e.
  • Tissue factor was captured using immobilized antibodies to TF and then relipidated with 30:70 mixture of phosphatidyl serine and phosphatidyl choline.
  • TF activity was measured by adding calcium (5 mM), clotting factors Vila (1 nM) and X (150 nM), and measuring the subsequent rate of factor Xa generation.
  • Figure 3 is a graph of TF activity in human plasma treated with an antibody to alt-hTF.
  • Figures 4A and 4B are graphs of recombinant alt-hTF activity in two assay systems.
  • Tissue factor compositions Membrane bound human tissue factor (“TF") is a polymer of 263 amino acids. At one end of the polymer is an amino (“NH 2 ”) group commonly referred to in the art as the amino (or “N”)-terminus. At the other end of the polymer is a carboxyl (“COOH”) group commonly referred to in the art as the carboxyl (or “C”)-terminus. The region between the N- and C- termini is commonly referred to as the "internal” region.
  • the schematic structure of the TF internal region is shown in Figure 1. Truncated soluble versions of membrane bound TF are typically from 1-3 (i.e., amino acids 1, 2 or 3) to 218-219, although a truncated version may include additional residues of the transmembrane region from 219-241.
  • the extracellular domain of TF is composed of two fibronectin type III domains, both of which contribute to binding of coagulation factor Vila. All four Vila structural domains (alpha- carboxyglutamic acid (Gla), EGF1, EGF2 and protease) make TF contacts and, therefore, the total TF-VIIa intermolecular contact area is large (1800
  • the dimensions of the entire TF:VIIa complex are about 115 A in length and 40-50 A in diameter.
  • Most of the molecular interactions seen in the TF:VIIa crystal structure are in agreement with mutational and biochemical analysis of TF: Vila and with binding residues identified from natural VII mutants, such as Arg 79 in the VIIa-EGFl domain.
  • the contacts provided by the Vila light chain, particularly the EGF1 domain, are the main contributors to the binding energy (approximately 6.6-9.6 kcal/mol) and also
  • Vila protease domain are less important with respect to energetic contribution (2.3-3.1 kcal/mol), but have a critical role in transmitting allosteric changes within the VII protease domain leading to enhanced catalytic activity.
  • the contact residues are mainly located on two adjacent alpha-helical stretches in the Vila protease domain: Vila Arg 134
  • chymotrypsinogen numbering scheme used throughout contacts TF-Asp 44 and TF-Trp 45, and Vila-Met 164 of the adjacent alpha-helix contacts TF- Phe 76 and TF-Tyr 94.
  • the Vila active site is located about 80 A above the membrane surface.
  • Factors VII, IX, X, protein C and prothrombin are anchored to the phospholipid surface via their Gla domains, which contains
  • the VIIa-Gla domain has three hydrophobic residues at positions 4, 5 and 8 (Phe 4, Leu 5, Leu 8).
  • coagulation factors IX, X and protein C suggest that occupancy of the Ca 2+ - binding sites induces the surface exposure of these residues (in IX the residues are at position 6, 9 and 10) which then engage in hydrophobic interactions with the phospholipid layer.
  • Vila the side chains of these three conserved hydrophobic residues point away from the rest of the Gla domain in a direction approximately perpendicular to the string of 6 calcium ions. Therefore, in analogy to the proposed function of the homologous residues in other coagulation enzymes, these amino acids may anchor Vila to membranes by insertion into the outer phospholipid layer.
  • Mutational changes at TF residues Lys 165 and Lys 166 revealed another region of TF that is important for enzymatic activity of the TF:VIIa complex. These two lysine residues are part of a surface region that interacts with substrates and is located outside the TF-VIIa interface area. The main region is composed of 7 residues (Tyr 157, Lys 159, Ser 163, Gly 164, Lys 165, Lys 166 and Tyr 185) forming a continuous surface patch of about 500
  • This substrate recognition region which may further extend to the Vlla- Gla domain, contacts the Gla domains of substrates X and IX. It may also interact with the EGF-1 domain of substrates as suggested by the impaired activation of two naturally occurring IX-EGF1 domain variants, Gly48Arg and Gly48Val, and by IX-EGF1 domain swap experiments.
  • This TF- substrate contact site may serve to properly align X and IX with respect to TF:VIIa complex allowing the formation of productive ternary TF: Vila: substrate complexes.
  • anti-TF antibodies that bind to this region potently inhibit activation of substrates X and IX.
  • the TF-mediated enhancement of Vila proteolytic activity is the aggregate of TF's interaction with substrate, the immobilization of Vila onto the cell surface and the proper positioning of the Vila protease domain.
  • TF also induces allosteric effects at the Vila catalytic center that contribute to an increase in Vila enzymatic activity.
  • the overall conformation of the SI recognition pocket as well as the position of the catalytic triad residues (His 57, Asp 102, Ser 195) in Vila is very similar to other serine proteases, such as IXa and Xa.
  • the Xa inhibitor, DX-9065a which selectively binds to the Xa active site, demonstrates that differences in the S2-S4 region can be exploited to design enzyme-specific reversible inhibitors.
  • the binding of VII to TF and its subsequent conversion to Vila is believed to constitute the initial proteolytic process in coagulation.
  • a new form of human tissue factor has been discovered. This form of human tissue factor circulates in plasma. Sequence analysis indicates that it is a soluble form of human tissue factor and apparently results from alternative splicing of the primary RNA transcript.
  • SEQ ID NO:l is the carboxyl-terminus of naturally occurring circulating alternatively spliced human tissue factor, amino acid residues 167 to 206, referred to herein as "alt-hTF.” This 40 amino acid sequence is unique to alt-hTF.
  • SEQ ID NO:2 is the cDNA sequence encoding amino acid residues 167 to 206 of alt-hTF.
  • the stop codon is underlined.
  • SEQ ID NO:l and SEQ ID NO:2 are useful in screening of patient samples for the presence of the normal alt-hTF protein, using hybridization assays of patient samples, including blood and tissues, as well as using the methods and reagents described in the examples. Screening can also be accomplished using antibodies, typically labeled with a fluorescent, radioactive, or enzymatic label, or by isolation of target cells and screening for clotting activity, as described in the examples. Typically, one would screen for expression on either a qualitative or quantitative basis, and for expression of functional alt-hTF.
  • Hybridization Probes Reaction conditions for hybridization of an oligonucleotide probe or primer to a nucleic acid sequence vary from oligonucleotide to oligonucleotide, depending on factors such as oligonucleotide length, the number of G and C nucleotides, and the composition of the buffer utilized in the hybridization reaction.
  • Moderately stringent hybridization conditions are generally understood by those skilled in the art as conditions approximately 25 °C below the melting temperature of a perfectly base-paired double- stranded DNA. Higher specificity is generally achieved by employing incubation conditions having higher temperatures, in other words more stringent conditions. In general, the longer the sequence or higher the G and C content, the higher the temperature and/or salt concentration required.
  • hybridization conditions for oligonucleotide probes and primers in great detail, including a description of the factors involved and the level of stringency necessary to guarantee hybridization with specificity.
  • the preferred size of a hybridization probe is from 10 nucleotides to
  • the probe should be from 20 to 10,000 nucleotides. Smaller nucleotide sequences (20-100) lend themselves to production by automated organic synthetic techniques. Sequences from 100-10,000 nucleotides can be obtained from appropriate restriction endonuclease treatments. The labeling of the smaller probes with the relatively bulky chemiluminescent moieties may in some cases interfere with the hybridization process.
  • Antibodies for Diagnostic or Therapeutic Use Antibodies to alt-hTF can also be generated that are useful in detection, characterization or isolation of alt-hTF, as well as for modifying alt-hTF protein activity, in most cases, through inhibition of clotting. Antibodies are generated by standard techniques, using alt-hTF. Since the proteins exhibit high evolutionary conservation, it may be advantageous to generate antibodies to the protein of a different species of origin than the species in which the antibodies are to be tested or utilized, looking for those antibodies that are immunoreactive with the most evolutionarily conserved regions.
  • Antibodies are typically generated by immunization of an animal using an adjuvant such as Freund's adjuvant in combination with an immunogenic amount of the protein administered over a period of weeks in two to three week intervals, then isolated from the serum, or used to make hybridomas that express the antibodies in culture. Because the methods for immunizing animals yield antibody that is not of human origin, the antibodies could elicit an adverse effect if administered to humans. Methods for "humanizing" antibodies, or generating less immunogenic fragments of non-human antibodies, are well known.
  • a humanized antibody is one in which only the antigen-recognized sites, or complementarity-determining hypervariable regions (CDRs) are of non-human origin, whereas all framework regions (FR) of variable domains are products of human genes.
  • variable region DNA of a selected animal recombinant anti-idiotypic ScFv is sequenced by the method of Clackson, T., et al., 1991 Nature. 352:624-688. Using this sequence, animal CDRs are distinguished from animal framework regions (FR) based on locations of the CDRs in known sequences of animal variable genes.
  • the CDRs are grafted onto human heavy chain variable region framework by the use of synthetic oligonucleotides and polymerase chain reaction (PCR) recombination. Codons for the animal heavy chain CDRs, as well as the available human heavy chain variable region framework, are built in four (each 100 bases long) oligonucleotides. Using PCR, a grafted DNA sequence of 400 bases is formed that encodes for the recombinant animal CDR/human heavy chain FR protection.
  • PCR polymerase chain reaction
  • the immunogenic stimulus presented by the monoclonal antibodies so produced may be further decreased by the use of Pharmacia's (Pharmacia LKB Biotechnology, Sweden) "Recombinant Phage Antibody System” (RPAS), which generates a single-chain Fv fragment (ScFv) that incorporates the complete antigen-binding domain of the antibody.
  • RPAS Recombinant Phage Antibody System
  • ScFv single-chain Fv fragment
  • antibody variable heavy and light chain genes are separately amplified from the hybridoma mRNA and cloned into an expression vector.
  • the heavy and light chain domains are co-expressed on the same polypeptide chain after joining with a short linker DNA that codes for a flexible peptide.
  • the antibodies can be formulated in standard pharmaceutical carriers for administration to patients in need thereof. These include saline, phosphate buffered saline, and other aqueous carriers, and liposomes, polymeric microspheres and other controlled release delivery devices, as are well known in the art.
  • the antibodies can also be administered with adjuvant, such as muramyl dipeptide or other materials approved for use in humans (Freund's adjuvant can be used for administration of antibody to animals).
  • Antibodies can also be immobilized as discussed below.
  • the levels of alt-hTF can be measured in plasma using a simple antibody immunoassay, or the mRNA can be measured using standard RT-PCR or other nucleotide based diagnostic assays.
  • an antibody that differentiates between native membrane bound TF or a truncated form thereof, and the alt-hTF This can be obtained by immunizing with the alt-hTF, alone or conjugated to a carrier, with or without adjuvant, then removing antibody that is produced that binds to the membrane bound TF using for example a column having membrane bound TF immobilized thereon.
  • a fragment of the alt- hTF derived from the last forty amino acids of the protein is used for immunization to generate alt-hTF antibodies.
  • alt-hTF levels of alt-hTF will vary with condition and disorder. Accordingly, conditions such as predisposition to clot will be predictable based on the levels of alt-hTF in the plasma. Normal levels are in the picomolar range. It is expected that the levels will be elevated in individuals having thrombotic disorders such as DVT. III. Therapeutic methods targeting alt-hTF
  • TF is a membrane-anchored cell-surface protein that initiates coagulation when blood contacts damaged tissue.
  • granulocytes were associated with thrombi in damaged jugular veins and were shown to be the source of thrombus associated TF activity.
  • u-PAR urokinase receptor
  • the TF-VIIa complex initiates coagulation and intracellular signaling. It also alters gene expression and promotes metastasis in murine models.
  • Various mechanisms, all involving active Vila, have been proposed to account for these varied responses. Accordingly, one should be able to treat clotting or the disorders discussed above, by blocking the activity, or removal of, the alt-hTF, using standard techniques to obtain suitable antibodies or other compounds specific for the alt-hTF, or removal techniques such as a column or filter having immobilized therein on antibody specifically immunoreactive with the alt- hTF. IV.
  • alt-hTF Designing or screening for drugs modifying or altering the extent of alt-hTF function or expression alt-hTF is useful as a target for compounds that turn on, or off, or otherwise regulate clotting or other disease processes mediated by alt-hTF.
  • the assays described in the examples clearly provide routine methodology by which a compound can be tested.
  • the in vitro studies of compounds that appear to inhibit alt-hTF are then confirmed by animal testing.
  • GPIIb/IIIa antagonists ADP antagonists such as clopidogrel, low-molecular weight heparins, and direct thrombin inhibitors).
  • ADP antagonists such as clopidogrel, low-molecular weight heparins, and direct thrombin inhibitors.
  • New research is leading to the next generation of antithrombotic compounds such as direct coagulation FVIIa inhibitors, tissue factor pathway inhibitors, gene therapy, and orally active direct thrombin inhibitors and coagulation Factor Xa (FXa) inhibitors.
  • Animal models of thrombosis have played a crucial role in discovering and validating novel drug targets, selecting new agents for clinical evaluation, and providing dosing and safety information for clinical trials. In addition, these models have provided valuable information regarding the mechanisms of these new agents and the interactions between antithrombotic agents that work by different mechanisms.
  • Genetic models have also been used in thrombosis/hemostasis research and pharmacology, for example, gene- therapy for hemophilia, is an example of how animal models have aided in the development of the therapies that are now being evaluated clinically.
  • Studies based on inhibition of clotting are predictive for indirect effects of alteration of alt-hTF binding
  • Assays for testing compounds for useful activity can be based solely on interaction with the alt-hTF protein.
  • the assays can be based on interaction with the gene sequence encoding the alt-hTF protein, or the regulatory sequences directing expression of the alt-hTF protein.
  • antisense that binds to the regulatory sequences, and/or to the protein encoding sequences can be synthesized using standard oligonucleotide synthetic chemistry.
  • the antisense can be stabilized for pharmaceutical use using standard methodology (encapsulation in a liposome or microsphere; introduction of modified nucleotides that are resistant to degradation or groups which increase resistance to endonucleases, such as phosphorothiodates and methylation), then screened initially for alteration of alt-hTF activity in transfected or naturally occurring cells which express alt- hTF, then in vivo in laboratory animals.
  • the antisense would inhibit expression.
  • sequences which block those sequences which "turn off" synthesis can also be targeted.
  • the alt-hTF protein for study can be isolated from either naturally occurring cells or cells which have been genetically engineered to express the alt-hTF protein, as described in the examples above.
  • Drug design using computational chemistry and molecular modeling Medicinal chemistry is an interdisciplinary approach used to design small molecules, such as organic chemicals or peptides, for use as therapeutic agents.
  • Medicinal chemists use a variety of technology platforms to discover and design drugs. These include combinatorial chemistry, computational chemistry, molecular modeling, high-throughput screening (HTS), enzymology, and pharmacology. The goal is to identify portions of a molecule responsible for particular activities, such as receptor binding or protein interaction. These properties can then be exploited to rationally design more effective drugs. Based on the structure and properties of a lead drug candidate, combinatorial chemists synthesize a series of closely related analogs.
  • Computational chemistry tools are then used to simulate the interactions of structural elements with macro-molecules, such as receptors, in order to correlate structure with activity.
  • Computational chemistry tools include tools for 3-D structure analysis, quantitative structure-activity relationship analysis, and comparative molecular field analysis, among others.
  • Several companies market software and services to help speed drug discovery and lead optimization programs. For example, Tripos Inc., St. Louis, produces a variety of "chemically intelligent" modeling and analysis tools through its discovery software program. Bio Balance, New York, is an example of a company that does computer modeling of proteins for drug design.
  • NMR nuclear magnetic resonance
  • This information usually resides in major, world-accessible databases including the Brookhaven Protein Data Bank for protein structures, the Nucleic Acids DataBase at Rutgers University for DNA structures, and the Cambridge Crystallographic Data Centre (CCDC) for small molecule (nonprotein/DNA/RNA) structures.
  • CCDC Cambridge Crystallographic Data Centre
  • investigators may dissect the intricate features of a molecule's structure or examine potential structural changes due to changes in the atomic or molecular composition of the molecule or macromolecule.
  • Three- dimensional structural analyses give the ability to examine the spatial, electrostatic, hydrophilic/hydrophobic, potential bonding, or the relationships of the substitute residue with neighboring residues on the same or separate chains.
  • Homology modeling has been very important these last few years, as researchers in academia and the pharmaceutical industry seek model structures for proteins whose crystal structures have not yet been solved. Homology modeling is also referred to as "comparative modeling” and "knowledge-based modeling.” It is essentially the theoretical creation of a structure using structural elements borrowed from another protein within the same protein family (usually based on primary sequence and/or secondary structure features) whose crystal structure is known. The process involves alignment of the two sequences, usually performed by any of a number of bioinformatics tools. The result of this alignment is then fed into a homology modeling application, which uses the known crystal structure and the alignment to construct a "draft" (preliminary) structure for the "structureless" protein.
  • Docking modeling is used to better understand and model novel protein-protein and protein-ligand interactions (that is, receptor and ligand binding). This provides an avenue to examine and model receptor sites and assess potential ligands (drugs) abd receptor-ligand associations. These techniques allow one to examine binding specificity and decipher the details of the atomic interactions involved in molecular recognition and catalysis.
  • Some of the more widely used docking programs include AutoDock (Oxford Molecular Group), DOCK (Molecular Design Institute-UC San Francisco), FTDOCK (Biomolecular Modeling Laboratory), INSIGHT II (MSI), SYBYL/FLEXIDOCK (Tripos) and MidasPlus (Computer Graphics Laboratory, UC San Francisco).
  • Modeling programs that utilize molecular dynamics function include HyperChem (Hypercube), INSIGHT II/Discover (MSI), AMBER, CAChe (OMG), SYBYL (Tripos), Alchemy 2000 (Tripos), Spartan (Wavefunction).
  • Molecular mechanics emphasizes the potential energy of the molecule as a function of its component atoms, bonds and their angles, and charges — in general, the entire macromolecular environment. This approach attempts to calculate an energy potential for the entire molecule.
  • Structure assignment is based on the assumption that the structure with the lowest energy potential represents a best fit for the molecule's structure as it exists in nature.
  • Modeling programs that utilize molecular mechanical methods include HyperChem (Hypercube), SCULPT (ISI), SYBYL (Tripos), INSIGHT II (MSI), AMBER, CAChe (OMG).
  • Quantum mechanics methods of "structure” determination are based upon the electronic makeup of a molecule. Electron distribution is defined by one of many quantum theories; the most widely known and used is the molecular orbital theory.
  • Modeling programs that utilize quantum mechanical methods include Cerius 2 , INSIGHT II (MSI), HyperChem (Hypercube), and SYBYL (Tripos). Many modeling applications make use of several mathematical methods; for example, Mac/PC/UNIX SPARTAN (Wavefunction), SYBYL (Tripos), and INSIGHT II (MSI) use a combination of molecular mechanics, quantum mechanical, and/or molecular dynamics methods. Multi-routine programs such as Cerius (Tripos), INSIGHT II (MSI), Look/GeneMine (MAG), MidasPlus (Computer Graphics Lab), and SYBYL (Tripos) are especially important to researchers performing homology modeling and docking, where various kinds of computational routines are utilized in the model-building process. Such a process incorporates all physicochemical properties into the computational equation to derive the best thermodynamically stable structure, a structure that should depict a functional molecule.
  • Computer modeling technology allows visualization of the three- dimensional atomic structure of a selected molecule and the rational design of new compounds that will interact with the molecule.
  • the three- dimensional construct typically depends on data from x-ray crystallographic analyses or NMR imaging of the selected molecule.
  • the molecular dynamics require force field data.
  • the computer graphics systems enable prediction of how a new compound will link to the target molecule and allow experimental manipulation of the structures of the compound and target molecule to perfect binding specificity. Prediction of what the molecule- compound interaction will be when small changes are made in one or both requires molecular mechanics software and computationally intensive computers, usually coupled with user-friendly, menu-driven interfaces between the molecular design program and the user.
  • Nucleic acid molecules containing the 5' regulatory sequences of the TF gene can be used to regulate or inhibit gene expression in vivo.
  • Vectors including both plasmid and eukaryotic viral vectors, may be used to express a particular recombinant 5' flanking region-gene construct in cells depending on the preference and judgment of the skilled practitioner (see, e.g.,
  • nucleic acid sequences in vivo (see, e.g., Mulligan, 1993 Science. 260, 926-932; United States Patent No. 4,980,286; United States Patent No. 4,868,116; incorporated herein by reference).
  • Delivery systems are available in which nucleic acid is encapsulated in cationic liposomes that can be injected intravenously into a mammal. This system has been used to introduce DNA into the cells of multiple tissues of adult mice, including endothelium and bone marrow (see, e.g., Zhu et al., 1993 Science 261, 209-211.
  • the 5' flanking sequences of the TF gene can also be used to inhibit the expression of alt-hTF.
  • an antisense RNA of all or a portion of the 5' flanking region of the TF gene can be used to inhibit expression of alt-hTF in vivo.
  • Expression vectors e.g., retroviral expression vectors
  • DNA containing all or a portion of the sequence of the 5' flanking region of the TF gene can be inserted into an appropriate expression vector so that upon passage into the cell, the transcription of the inserted DNA yields an antisense RNA transcript that is complementary to the mRNA transcript of the alt-hTF protein normally found in the cell.
  • This antisense RNA transcript of the inserted DNA can then base-pair with the normal mRNA transcript found in the cell and thereby prevent the mRNA from being translated. It is of course necessary to select sequences that are downstream from the transcriptional start sites for the TF gene to ensure that the antisense RNA contains sequences complementary to those present in the mRNA.
  • Antisense RNA can be also generated in vitro, and then inserted into cells.
  • Oligonucleotides can be synthesized on an automated synthesizer (e.g., Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B).
  • an automated synthesizer e.g., Model 8700 automated synthesizer of Milligen-Biosearch, Burlington, MA or ABI Model 380B.
  • antisense deoxyoligonucleotides have been shown to be effective in inhibiting gene transcription and viral replication (see e.g., Zamecnik et al., 1978 Proc. Natl. Acad. Sci. USA 75, 280-284; Zamecnik et al., 1986 Proc. Natl. Acad. Sci.. 83, 4143-4146; Wickstrom et al., 1988 Proc. Natl. Acad. Sci. USA 85, 1028-1032; Crooke, 1993 FASEB J.
  • antisense oligonucleotides Inhibition of expression of a gene by antisense oligonucleotides is also possible if the antisense oligonucleotides contain modified nucleotides (see, e.g., Offensperger et. al., 1993 EMBO J. 12, 1257-1262 (antisense phosphorothioate oligodeoxynucleotides); Rosenberg et al., PCT WO 93/01286 (synthesis of sulfurthioate oligonucleotides);
  • RNA interference see, e.g., Sharp, Genes & Development, 2001. 15:485-490.
  • Double-stranded RNA molecules can be synthesized in vitro and then introduced into living cells (see, e.g., Donze et al., 2002 Nucleic Acid Research, 30:e46) or synthesized from a DNA template that was stably incorporated into cells (see, e.g., Sui et al., 2002 Proc. Natl. Acad. Sci. USA 99:5515-5520).
  • Double-stranded RNA molecules have been shown to inhibit HIV-1 infection (see Novina et al., 2002, Nature Medicine, 8:681-686) and expression of the full-length tissue factor (see Holen et al., Nucleic Acid Research 2002, 30:1757-1766).
  • double-stranded RNA molecules containing the region unique to alt-hTF mRNA, i.e. the site of splicing of exon 4 to exon 6 may be used to selectively inhibit expression of alt-hTF protein in vivo.
  • sequences of the 5' flanking region of the TF gene can also be used in triple helix (triplex) gene therapy. Oligonucleotides complementary to gene promoter sequences on one of the strands of the DNA have been shown to bind promoter and regulatory sequences to form local triple nucleic acid helices which block transcription of the gene (see, e.g., 1989 Maher et al., Science 245, 725-730; Orson et al., 1991 Nucl. Acids Res. 19, 3435- 3441; Postal et al., 1991 Proc. Natl. Acad. Sci.
  • oligonucleotides should generally be greater than 14 nucleotides in length to ensure target sequence specificity (see, e.g., Maher et al., 1989; Grigoriev et al., 1992). Also, many cells avidly take up oligonucleotides that are less than 50 nucleotides in length (see e.g., Orson et al., 1991; Holt et al., 1988 Mol. Cell. Biol. 8, 963-973; Wickstrom et al., 1988 Proc. Natl. Acad. Sci. USA 85, 1028-1032).
  • a free amine can be introduced to a 3' terminal hydroxyl group of oligonucleotides without loss of sequence binding specificity (Orson et al., 1991). Furthermore, more stable triplexes are formed if any cytosines that may be present in the oligonucleotide are methylated, and also if an intercalating agent, such as an acridine derivative, is covalently attached to a 5' terminal phosphate (e.g., via a pentamethylene bridge); again without loss of sequence specificity (Maher et al., 1989; Grigoriev et al., 1992).
  • an intercalating agent such as an acridine derivative
  • oligonucleotides are well known in the art and synthetic oligonucleotides are now commercially available. Such methods can range from standard enzymatic digestion followed by nucleotide fragment isolation (see e.g., Sambrook et al., Chapters 5, 6) to purely synthetic methods, for example, by the cyanoethyl phosphoramidite method using a Milligen or Beckman System lPlus DNA synthesizer (see also, Ikuta et al., in Ann. Rev. Biochem.
  • DNA sequences of the 5' flanking region of the TF gene described herein can be used to design and construct oligonucleotides including a DNA sequence consisting essentially of at least 15 consecutive nucleotides, with or without base modifications or intercalating agent derivatives, for use in forming triple helices specifically within the 5' flanking region of the TF gene in order to inhibit expression of the gene.
  • enhancers or multiple copies of the regulatory sequences may be advantageous to insert enhancers or multiple copies of the regulatory sequences into an expression system to facilitate screening of methods and reagents for manipulation of expression.
  • Molecules with a given function, catalytic or ligand-binding can be selected for from a complex mixture of random molecules in what has been referred to as "in vitro genetics" (Szostak, TIBS 19:89, 1992).
  • In vitro genetics One synthesizes a large pool of molecules bearing random and defined sequences and subjects that complex mixture, for example, approximately 10 15 individual sequences in 100 ⁇ g of a 100 nucleotide RNA, to some selection and enrichment process.
  • Ellington and Szostak (1990) estimated that 1 in 10 10 RNA molecules folded in such a way as to bind a given ligand. DNA molecules with such ligand-binding behavior have been isolated (Ellington and Szostak, 1992; Bock et al, 1992).
  • Compounds that are effective for blocking binding of the alt-hTF can also consist of fragments of the alt-hTF protein, expressed recombinantly and cleaved by enzymatic digest or expressed from a sequence encoding a peptide of less than the full length alt-hTF protein. These will typically be soluble proteins, i.e., not including the transmembrane and cytoplasmic regions, although smaller portions determined in the assays described above to inhibit or compete for binding to the alt-hTF protein can also be utilized. It is a routine matter to make appropriate alt-hTF protein fragments, test for binding, and then utilize them. The preferred fragments are of human origin, in order to minimize potential immunological response.
  • the peptides can be as short as five to eight amino acids in length and are easily prepared by standard techniques. They can also be modified to increase in vivo half-life, by chemical modification of the amino acids or by attachment to a carrier molecule or inert substrate. Based on studies with other peptide fragments blocking alt-hTF binding, the IC50, the dose of peptide required to inhibit binding by 50%, ranges from about 50 ⁇ M to about 300 ⁇ M, depending on the peptides. These ranges are well within the effective concentrations for the in vivo administration of peptides, based on comparison with the RGD- containing peptides, described, for example, in U.S. Patent No.
  • the peptides can also be conjugated to a carrier protein such as keyhole limpet hemocyanin by its N-terminal cysteine by standard procedures such as the commercial Imject kit from Pierce Chemicals or expressed as a fusion protein, which may have increased efficacy.
  • a carrier protein such as keyhole limpet hemocyanin by its N-terminal cysteine by standard procedures such as the commercial Imject kit from Pierce Chemicals or expressed as a fusion protein, which may have increased efficacy.
  • the peptides can be prepared by proteolytic cleavage of the alt-hTF protein, or, preferably, by synthetic means. These methods are known to those skilled in the art. An example is the solid phase synthesis described by J. Merrifield, 1964 J. Am. Chem. Soc. 85, 2149, used in U.S. Patent No.
  • the peptide can also be administered as a pharmaceutically acceptable acid- or base- addition salt, formed by reaction with inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid, and organic acids such as formic acid, acetic acid, propionic acid, glycolic acid, lactic acid, pyruvic acid, oxalic acid, malonic acid, succinic acid, maleic acid, and fumaric acid, or by reaction with an inorganic base such as sodium hydroxide, ammonium hydroxide, potassium hydroxide, and organic bases such as mono-, di-, trialkyl and aryl amines and substituted ethanolamines.
  • inorganic acids such as hydrochloric acid, hydrobromic acid, perchloric acid, nitric acid, thiocyanic acid, sulfuric acid, and phosphoric acid
  • organic acids such as formic acid, acetic acid, propionic acid
  • Peptides containing cyclopropyl amino acids, or amino acids derivatized in a similar fashion can also be used. These peptides retain their original activity but have increased half-lives in vivo. Methods known for modifying amino acids, and their use, are known to those skilled in the art, for example, as described in U.S. Patent No. 4,629,784 to Stammer. V. Pharmaceutical compositions
  • Peptides are generally active when administered parenterally in amounts above about 1 ⁇ g/kg of body weight. Based on extrapolation from other proteins, for treatment of most inflammatory disorders, the dosage range will be between 0.1 to 70 mg/kg of body weight. This dosage will be dependent, in part, on whether one or more peptides are administered.
  • the peptide sequences present in the C-terminal region of alt-hTF should be the most optimal. Because this sequence has not been described in other proteins, this would be a unique target to inhibit binding alt-hTF to platelets, thereby uniquely inhibiting the activity of alt-hTF.
  • binding activity Compounds that alter alt-hTF protein activity and/or binding (referred to generally herein as "binding activity") are preferably administered in a pharmaceutically acceptable vehicle.
  • suitable pharmaceutical vehicles are known to those skilled in the art.
  • the compound will usually be dissolved or suspended in sterile water or saline.
  • the compound will be incorporated into an inert carrier in tablet, liquid, or capsular form.
  • Suitable carriers may be starches or sugars and include lubricants, flavorings, binders, and other materials of the same nature.
  • the compounds can also be administered locally by topical application of a solution, cream, gel, or polymeric material (for example, PluronicTM, BASF).
  • the compound may be administered in liposomes or microspheres (or microparticles).
  • Methods for preparing liposomes and microspheres for administration to a patient are known to those skilled in the art.
  • U.S. Patent No. 4,789,734 describes methods for encapsulating biological materials in liposomes. Essentially, the material is dissolved in an aqueous solution, the appropriate phospholipids and lipids added, along with surfactants if required, and the material dialyzed or sonicated, as necessary.
  • a review of known methods is by G. Gregoriadis, Chapter 14. "Liposomes", Drug Carriers in Biology and Medicine pp. 287-341 (Academic Press, 1979).
  • Microspheres formed of polymers or proteins are well known to those skilled in the art, and can be tailored for passage through the gastrointestinal tract directly into the bloodstream. Alternatively, the compound can be incorporated and the microspheres, or composite of microspheres, implanted for slow release over a period of time, ranging from days to months. See, for example, U.S. Patent No. 4,906,474, 4,925,673, and 3,625,214. VI. Removal of alt-hTF from patients or patient samples
  • the antibodies to alt-hTF protein can be used to remove alt-hTF from patient blood, by immobilizing the alt-hTF antibodies on a suitable substrate, such as the cellulose membrane of a dialysis unit, using conventional coupling, for example, using carboimide. The patient's blood is then dialyzed through the unit.
  • Example 1 Characterization of tissue factor activity in plasma. An acellular form of tissue factor that appears to circulate in human plasma in a latent form has been identified. Upon shear-induced binding to platelets, this protein exhibits enzymatic activity and, therefore, may serve as a potential trigger for initiating or propagating thrombosis. Following highspeed centrifugation of already platelet-poor plasma, the activity remained in the bulk aqueous phase indicating that blood-borne TF is soluble in an aqueous medium. In this regard, we note that alt-hTF is extracted from bacteria simply by osmotic shock.
  • Citrated blood was processed as follows. After centrifugation at 200xG for 15 minutes, the cell pellet was discarded and the supernatant was mixed 1:1 with Tris-buffered saline (TBS) and is referred to as the "starting material.” Nine mL of the starting material was removed and centrifuged at 260,000xG for 4 hours. Following high-speed centrifugation, 4 distinct layers were evident and their volume fractions were estimated: an upper lipid phase (17% of volume), a bulk-aqueous phase (52%), a lower viscous phase (23%) and a pellet (3%). Samples from the starting material and each of the three upper phases were extracted and assayed for TF activity using a standardized protocol. In one experiment, the pellet was resuspended in 4.5mL of TBS and also assayed for TF activity.
  • TBS Tris-buffered saline
  • agrose beads covalently linked to a rabbit anti- human TF antibody (0.5 mg antibody per mL of agarose).
  • the beads were washed 5 times with hepes-buffered saline (HBS) and the bound antigen was eluted with 6M guanidine dissolved in HBS.
  • HBS hepes-buffered saline
  • the eluted protein was collected and incubated with phospholipids (30% phosphatidyl serine, 70% phosphatidyl choline; 75 ⁇ M total) and n-octyl- ⁇ -D-glucopyranoside.
  • the samples were dialyzed to remove the detergent and allow the formation of lipid vesicles in the presence of the eluted protein.
  • the dialyzed samples were supplemented with calcium (5mM), coagulation factor Vila (1 nM) and coagulation factor X (150 nM), and the rate of appearance of FXa was measured using a standard assay. The results are shown in Figure 2.
  • the TF activity associated with the starting material was 158.5 pM-FXa/min.
  • TF activity measured in each phase was 45.7 pM-FXa/min for the lipid phase, 138.9 pM-FXa/min for the bulk aqueous phase, 554.7 pM-FXa/min for the lower viscous phase, and 40 pM-FXa/min for the resuspended pellet. Background levels were approximately 20 pM-FXa/min.
  • TF mRNA was examined in a human cell line HL-60 using RT-PCR.
  • a pair of primers designated "Forward” and “Reverse” yielded two bands.
  • the size of the larger band was approximately 400 base pairs - as expected from the TF cDNA sequence.
  • the size of the smaller band was approximately 240 base pairs.
  • the smaller band was subcloned and sequenced. Sequencing results revealed a previously unknown splicing variant of TF mRNA in which exon 5 is absent and exon 4 is thus fused directly with exon 6. Of note, such a fusion creates a frameshift in the TF open reading frame.
  • the 770 base pair product was subcloned and sequenced.
  • the results of sequencing confirmed the existence of the TF mRNA spieces encoding an open reading frame with the initiation codon corresponding to bases 112-114, and a termination codon corresponding to bases 986-988 of the published TF cDNA sequence; GenBank accession number J02931.
  • the alternatively spliced hTF mRNA lacks exon 5 and thus contains a frameshift.
  • the open reading frame of the alt-hTF mRNA encodes a human tissue factor variant whose mature peptide comprises 206 amino acids.
  • Amino acids 1-166 are identical to those of the known membrane bound human TF; however, the remaining 40 amino acids at the carboxy- terminus diverge from the known TF amino acid sequence.
  • the carboxy-terminus of the alternatively spliced hTF contains a region with potential transmembrane properties.
  • Example 3 Expression of alt-hTF in various tissues.
  • the lengths of reported sequences of the three clones derived from a human lung cDNA library are 663, 780, and 747 base pairs, respectively. None of the three sequences encode a significantly larger non-interrupted stretch of amino acids in any of the three 5 '-3' frames. Although all three sequences contain a region corresponding to the site of alternative splicing, i.e., TCA GGA AAG AAA TAT TCT (SGKKYS) (SEQ ID NO:3), in all three sequences this region is not in frame with the sequence encoding human tissue factor protein.
  • the three clones corresponding to the above lung ESTs were obtained from the I.M.A.G.E. consortium and fully sequenced. None of the clones contained the complete alt-hTF open reading frame. However, sequencing results revealed that, like in membrane-bound TF mRNA, the long 3 '-untranslated end of the asHTF mRNA is entirely encoded by exon 6.
  • the length of the reported sequence of a clone derived from a primary human keratinocyte cDNA library (BF149254) is 356 base pairs. This sequence encodes a stretch of amino acids 72 through 166 of the membrane bound human tissue factor fused to the first 23 amino acids of the 40 amino acid carboxyl-terminus unique to the alternatively spliced hTF. This sequence is, therefore, a partial (incomplete) cDNA encoding an alternatively spliced human tissue factor molecule.
  • Example 4 Characterization of plasma depleted of the alt-hTF protein.
  • start plasma Frozen human plasma (300 mL) was thawed on ice and centrifuged at 3,000xG for 90 minutes. The supernatant (referred to as "start plasma") was passed at 4°C through a column loaded with Affigel-coupled antibodies to the last 10 amino acids at the carboxyl-terminus of the alt-hTF protein. A flow-through sample was collected after 250 mL of start plasma was passed through the column.
  • Samples described above were tested for tissue factor activity using a Xa chromogenic assay in a platelet-shear system.
  • Freshly drawn citrated blood was centrifuged at 135xG for 20 minutes at room temperature to prepare platelet rich plasma (PRP). Platelets were then pelleted by centrifuging PRP at 850xG for 12 minutes, washed twice in CGSa, and resuspended at 3x10 platelets/mL in modified Tyrode's Buffer.
  • 75 ⁇ L of the test samples i.e. start plasma and plasma-flow-through
  • 75 ⁇ L of the platelet suspension were mixed with 75 ⁇ L of the platelet suspension.
  • the plasma-platelet suspension was loaded into collagen-coated wells.
  • Example 5 Expression of the alt-hTF protein in bacteria.
  • the region encoding the entire mature peptide of the alternatively spliced hTF variant was amplified using RT-PCR and subcloned into pBAD/gHIA expression vector (Invitrogen Corporation). The sequence of this construct was verified, and the recombinant alternatively spliced hTF protein was produced in E. coli.
  • three additional amino acids i.e. Thr, Met, and Ala
  • the expressed protein was isolated from bacteria by osmotic shock, and the resultant osmotic shock fluid was concentrated via centrifugation in CentriconTM filter devices (Millipore). Presence of the desired protein in the concentrated osmotic shock fluid was verified by Western immunoblotting, and protein concentration was analysed using Bradford protein microassay. Three samples containing the recombinant alt-hTF protein were then prepared: a sample of the protein dissolved in HBS, a sample of the relipidated protein, and a sample of the protein incubated with phospholipid vesicles. Relipidation of the recombinant protein was carried out as follows.
  • 35 ⁇ g of protein in TBS were combined with n-octyl- ⁇ -D-glucopyranoside (final concentration - 125 mM) and a phospholipid mixture (PS:PC 30:70, final concentration - 75 ⁇ M) to the final volume of 0.5 mL.
  • the sample was placed on orbital mixer (set to slow) for 30 min at RT, transferred into a 0.5 mL dialysis cassette and dialyzed overnight versus 2 L of TBS.
  • Incubation of the recombinant protein with phospholipid vesicles was carried out as follows.
  • Example 6 Presence of the alt-TF protein in ex-vivo thrombi.

Abstract

On a identifié une nouvelle forme circulante de facteur tissulaire humain soluble. Cette nouvelle forme de facteur tissulaire humain semble provenir d'un épissage alternatif et est par conséquent désignée 'alt-hTF.'. On a détecté l'ARNm de Alt-hTF dans une lignée cellulaire, HL-60. On a cloné la région d'ADNc codant la totalité du cadre de lecture ouvert de alt-hTF. La séquence codant le peptide mature de alt-hTF a été exprimée dans des bactéries. alt-hTF est constitué par les 166 premiers acides aminés du TF fixé à la membrane et par une région unique de terminaisons-C de 40 acides aminés. alt-hTF semble être une cible utile pour des composés servant à inhiber la coagulation et à traiter des maladies associées à un niveau élevé de TF. Il peut également être utile en tant que cible pour des anticorps présentant une réaction sélective avec alt-hTF, de manière à le supprimer de la circulation afin de traiter la coagulation ou d'autres maladies associées à des niveaux élevés ou anormaux de TF, y compris des thromboses, des maladies cardio-vasculaires, DVT, DIC et, éventuellement, des cancers métastatiques.
PCT/US2002/027533 2001-08-30 2002-08-29 Facteur tissulaire circulant presentant un epissage alternatif WO2003020111A2 (fr)

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Cited By (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003066650A2 (fr) * 2002-02-07 2003-08-14 Hans Prydz Suppression post transcriptionnelle de l'expression par des arn a interferences courtes
WO2004094475A2 (fr) * 2003-04-22 2004-11-04 Euro-Celtique S.A. Anticorps se fixant au facteur tissulaire et leurs utilisations
WO2011144953A1 (fr) * 2010-05-18 2011-11-24 Diagon Kft. Procédure pour produire un facteur tissulaire humain recombinant
US9150658B2 (en) 2008-12-09 2015-10-06 Genmab A/S Human antibodies against tissue factor and methods of use thereof
US9168314B2 (en) 2010-06-15 2015-10-27 Genmab A/S Human antibody drug conjugates against tissue factor

Families Citing this family (5)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2008054822A2 (fr) * 2006-11-03 2008-05-08 Angiogen Llc Compositions comprenant un facteur tissulaire à épissure alternée (tf) et procédés d'utilisation
US20100297621A1 (en) * 2007-06-20 2010-11-25 University Of Utah Research Foundation Use of pre-mrna splicing in platelet cells for the diagnosis of disease
US9555109B2 (en) 2012-01-20 2017-01-31 University Of Cincinnati Method of inhibiting cell proliferation induced by alternatively spliced tissue factor by administering a monoclonal antibody
US10204181B1 (en) * 2015-07-10 2019-02-12 Omnisent LLC Systems and methods for modeling quantum structure and behavior
US10637583B2 (en) * 2015-07-10 2020-04-28 Omnisent, LLC Systems and methods for modeling quantum entanglement and performing quantum communication

Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5346991A (en) * 1991-06-13 1994-09-13 Genentech, Inc. Tissue factor mutants useful for the treatment of myocardial infarction and coagulopathic disorders

Family Cites Families (23)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US3625214A (en) 1970-05-18 1971-12-07 Alza Corp Drug-delivery device
US4244946A (en) 1979-06-11 1981-01-13 The Salk Institute For Biological Studies Water-soluble peptides affecting gonadal function
US4305872A (en) 1979-10-19 1981-12-15 Kenneth Wingrove Polypeptide derivatives
US4316891A (en) 1980-06-14 1982-02-23 The Salk Institute For Biological Studies Extended N-terminal somatostatin
US4792525A (en) 1982-08-04 1988-12-20 La Jolla Cancer Research Foundation Tetrapeptide
US4906474A (en) 1983-03-22 1990-03-06 Massachusetts Institute Of Technology Bioerodible polyanhydrides for controlled drug delivery
US4629784A (en) 1983-08-16 1986-12-16 The University Of Georgia Research Foundation, Inc. Synthesis of cyclopropane amino acids
US4980286A (en) 1985-07-05 1990-12-25 Whitehead Institute For Biomedical Research In vivo introduction and expression of foreign genetic material in epithelial cells
IL79289A (en) 1985-07-05 1992-01-15 Whitehead Biomedical Inst Introduction and expression of foreign genetic material into keratinocytes using a recombinant retrovirus
US4789734A (en) 1985-08-06 1988-12-06 La Jolla Cancer Research Foundation Vitronectin specific cell receptor derived from mammalian mesenchymal tissue
NL8720442A (nl) 1986-08-18 1989-04-03 Clinical Technologies Ass Afgeefsystemen voor farmacologische agentia.
NO892506L (no) 1988-06-17 1989-12-18 Sinai School Medicine Kloning og ekspresjon av human vevsfaktor.
CA1340977C (fr) 1988-11-15 2000-04-25 Monty Krieger Recepteur proteique capteur, ainsi que son anticorps
US5211947A (en) 1988-12-16 1993-05-18 Schering Corporation Method for lowering blood cholesterol levels with granulocyte-macrophage colony stimulating factor
JPH03290184A (ja) 1990-04-06 1991-12-19 Chugai Pharmaceut Co Ltd スカベンジャーレセプター産生動物細胞
JP3594318B2 (ja) 1990-08-27 2004-11-24 中外製薬株式会社 抗ヒトスカベンジャーレセプター抗体
JPH07501204A (ja) 1991-06-28 1995-02-09 マサチューセッツ インスティテュート オブ テクノロジー 局所的オリゴヌクレオチド療法
WO1993019166A1 (fr) 1992-03-23 1993-09-30 The University Of North Carolina At Chapel Hill Petits animaux modeles pour l'etude de metabolisme du cholesterol
US5585479A (en) 1992-07-24 1996-12-17 The United States Of America As Represented By The Secretary Of The Navy Antisense oligonucleotides directed against human ELAM-I RNA
US5746223A (en) 1996-10-11 1998-05-05 Williams; Kevin Jon Method of forcing the reverse transport of cholesterol from a body part to the liver while avoiding harmful disruptions of hepatic cholesterol homeostasis
US6429289B1 (en) 1994-06-23 2002-08-06 Massachusetts Institute Of Technology Class BI and CI scavenger receptors
US5652224A (en) 1995-02-24 1997-07-29 The Trustees Of The University Of Pennsylvania Methods and compositions for gene therapy for the treatment of defects in lipoprotein metabolism
US5925333A (en) 1995-11-15 1999-07-20 Massachusetts Institute Of Technology Methods for modulation of lipid uptake

Patent Citations (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US5346991A (en) * 1991-06-13 1994-09-13 Genentech, Inc. Tissue factor mutants useful for the treatment of myocardial infarction and coagulopathic disorders

Non-Patent Citations (1)

* Cited by examiner, † Cited by third party
Title
See also references of EP1429650A2 *

Cited By (12)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
WO2003066650A2 (fr) * 2002-02-07 2003-08-14 Hans Prydz Suppression post transcriptionnelle de l'expression par des arn a interferences courtes
WO2003066650A3 (fr) * 2002-02-07 2004-05-21 Hans Prydz Suppression post transcriptionnelle de l'expression par des arn a interferences courtes
WO2004094475A2 (fr) * 2003-04-22 2004-11-04 Euro-Celtique S.A. Anticorps se fixant au facteur tissulaire et leurs utilisations
WO2004094475A3 (fr) * 2003-04-22 2005-03-17 Euro Celtique Sa Anticorps se fixant au facteur tissulaire et leurs utilisations
JP2007525944A (ja) * 2003-04-22 2007-09-13 ユーロ−セルティーク エス．エイ． 組織因子抗体およびその使用
US7425328B2 (en) 2003-04-22 2008-09-16 Purdue Pharma L.P. Tissue factor antibodies and uses thereof
US7993644B2 (en) 2003-04-22 2011-08-09 Purdue Pharma L.P. Tissue factor antibodies and uses thereof
US9150658B2 (en) 2008-12-09 2015-10-06 Genmab A/S Human antibodies against tissue factor and methods of use thereof
US9714297B2 (en) 2008-12-09 2017-07-25 Genmab A/S Human antibodies against tissue factor and methods of use thereof
WO2011144953A1 (fr) * 2010-05-18 2011-11-24 Diagon Kft. Procédure pour produire un facteur tissulaire humain recombinant
US9168314B2 (en) 2010-06-15 2015-10-27 Genmab A/S Human antibody drug conjugates against tissue factor
US9492565B2 (en) 2010-06-15 2016-11-15 Genmab A/S Human antibody drug conjugates against tissue factor

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AU2006200669A1 (en) Claudin polypeptides

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